To date, numerous efforts have been made to fulfil the purpose of realizing quantity production of a useful protein by the use of the technique of gene recombination.
The technique of gene recombination for the quantity production of a useful protein basically comprises a host, a vector, and a gene coding a useful protein.
The hosts which are usable for this technique include procaryotes such as Escherichia coli and Bacillus subtilis and eucaryotes such as yeast, animal cell, and plant cell. In the selection of a host for a particular recombination, due consideration is paid to the characterization of a protein subjected to expression and the use for which the produced protein is intended.
As regards the vector to be used for the recombination, a large number of widely varying vectors have been developed to date. There are four basic functions which are required of a vector; (1) an ability to form an in vitro recombinant with a DNA which codes the protein aimed at, (2) an ability to attain growth in the cell of the host aimed at, (3) an ability to attain introduction in the cell of the host aimed at, and (4) ability to effect specific detection of a cell possessing a recombinant DNA. For the purpose of fulfilling quantity production of the useful protein, the vector is further required to possess these additional functions; (5) ability to possess a strong promoter and a terminator (DNA sequences concerning expression) and (6) an ability to possess a signal sequence (DNA sequences concerning secretion), for example.
A strong promoter is necessary for and indispensable to quantity production of a protein. The secretion of a mass-produced protein by a signal sequence is effective in preventing intracellular accumulation of a protein harmful to the host, precluding decomposition of a product by a protease in the cell, and simplifying and economizing the process of purification of a useful protein which has heretofore entailed expenditure of great labor and cost.
Owing to the advantages mentioned above, efforts are being continued in research and development of strong promoters and signal sequences excelling in efficiency of secretion.
In the case of Escherichia coli as a procaryote, a P.sub.L O.sub.L promoter for .lambda. phage, a lac promoter for lactose operon, a trp promoter for tryptophan operon, a lpp promoter and a signal sequence for an outer-membrane protein gene, and a lacUV5 promoter and a tac promoter as improved versions thereof have been developed. In the case of Bacillus subtilis, a penP for an enzyme penicillinase gene outside bacteria and a promoter and a signal sequence for an .alpha.-amylase have been developed.
In the case of yeasts as an eucaryote, promoters for a group of glycolytic enzymes have been demonstrated as effective for strong and over expression of proteins. For example, promoters and a-factors and signal sequence of such .alpha.-factor for genes such as 3-phosphoglycerate kinase (PGK), glyceraldehyde triphosphoric acid dehydrogenase (GLD), enolase (ENO), triose phosphoric acid isomerase (TPI), alcohol dehydrogenase (ADH), acidic phosphatase (PHO), and the galactose metabolic system (GAL), have been developed and put to use.
The number of cases of successful development of promoters usable for cells of higher animals is still small. Though promoters for early gene and late gene of the virus SV40 attaining satisfactory propagation in the cell of monkey, a promoter for an ICP gene of HSV, a promoter for an early gene of vaccinia virus, a promoter for a chicken .beta.-actin, a promoter for a human EF-1a gene, and an IgG H chain promoter have been developed, they are not fit for the purpose of quantity production of useful proteins.
As regards promoters which are usable for the technique of gene recombination using a plant as a host, a promoter for the 35S gene of a cauliflower mosaic virus, a promoter for a nopalin synthetic gene of a Ti plasmid, and an ORF12 promoter for a Ri plasmid have been developed. Again, these promoters are unfit for the purpose of over-production of useful proteins.
Recently, development of systems for secretionary production of useful proteins by the use of a mold particularly of genus Aspergillus has come to appear in literature. The secretionary production of such proteins as lipase and prochymosin by the use of a promoter for a glucoamylase gene of Aspergillus niger and a signal sequence has been realized, for example.
This statement does not necessarily mean that the use of systems capable of expression and secretion of mold of genus Aspergillus permits efficient and secretionary over production of all useful proteins. Thus, efforts are being continued in research and development of a system capable of more efficient expression and secretion.
Incidentally, a technique of gene recombination using a basidiomycete as a host remains yet to be established. The basidiomycetes include numerous useful fungi such as edible mushroom, fungi producing physiologically active substances, fungi capable of decomposing lignin and useful for biological pulping and biobleaching, and fungi decomposing cellulose and saccharifying lignous components. Attempts at improving and fortifying the characteristics of these fungi and breeding these fungi have been made heretofore with a method resorting to mating, a method resorting to acquisition of a variant, and a method resorting to cell fusion, for example. If a method for molecular breeding by the use of the technique of gene recombination is realized, it would allow easy acquisition of excellent strains.
Further, since the safety of using basidiomycetes for food, similar to that of Aspergilus oryzae (Koji-mould), has been already established, these basidiomycetes are highly useful as hosts for the production of proteins. An attempted use of a filamentous plasmid DNA occurring in the mitochondria of Lentinus edodes (Shiitake) and Pleurotus ostreatus (Hiratake) as a vector for a basidiomycete has been reported ("Iden [genetics]," vol. 42, No. 9, p. 20, Shokabo, 1988). It has been shown, however, that the filamentous plasmid DNA has problems such as lack of stability within the host and has not been perfected for practical use so far.
No promoter has been so far developed which is used effectively for basidiomycetes. Virtually no successful cloning of a gene for providing a promoter has yet been reported in literature, except for a report concerning a ligninase gene obtained by cloning with a microorganism of genus Phanerochaete chrysosporium. This gene is characterized by expressing ligninase by virtue of secondary metabolism and the extent of this expression is not appreciably large. Thus, the gene does not deserve to be called an effective promoter. In the circumstances, a desire has been expressed in the industry to develop a promoter and a signal sequence which are capable of effecting efficient secretion and expression of useful proteins with basidiomycete. The promoter and signal sequence thus yearned for are required to effect strong expression in a wide variety of hosts and possess a signal sequence for allowing secretion of a protein produced by the expression.
The Phenoloxidase which is a useful protein is such that the gene thereof, when introduced in a varying organism and expressed therein, can be utilized for biological pulping, biobleaching, decolorization of plant effluent, and pretreatment of wood in saccharification and can be used otherwise as a reagent for clinical tests.
The gene which codes this phenoloxidase has been developed and identified by the present inventors in accordance with a technique of cloning Coriolus hirsutus IFO 4917, i.e. a white-rot fungus [Japanese Patent Application 88-175,235 and 88-175,236].
A promoter and a signal sequence which are available for effecting expression, particularly secretionary expression, of this phenoloxidase gene, however, remain yet to be developed.
[Problem for Solution by the Invention]
The present inventors have pursued a diligent study with a view to fulfilling the demand for development of a promoter, a signal sequence, and a terminator capable of secretionary production of all useful proteins in large amounts, particularly the demand for development of such substances usable even with basidiomycetes. They have consequently succeeded in developing novel DNA's, i.e. a promoter, a signal sequence, and a terminator, concerning the expression and secretion, which attain secretionary production of phenoloxidase in a basidiomycete in a large amount.